Cytospectrofluorometric characterization of OPT-induced fluorescence in rat pinealocytes

Summary

o-Phthaldialdehyde (OPT) has been employed for the fluorescence histochemical demonstration of histamine. OPT-induced fluorescence with properties different from those of the histamine fluorophore was examined in cells of rat pineal, retina and endocrine pancreas. The fluorescence in the pinealocytes had two excitation maxima, at 370 and 430 nm, respectively. The corresponding emission maxima were at or below 410 nm and at 500 nm. Adjacent mast cells exhibited an identical fluorescence, suggesting that the OPT-reactive compound is released from the pinealocytes. The fluorescence in the retina was of orange colour and confined to the outer nuclear layer. The excitation maxima were at 380 and 410 nm, and the emission maximum was at 575 nm. A blue, OPT-induced fluorescence could be demonstrated in the pancreatic A₂-cells. Cytospectrofluorometric analysis revealed two excitation maxima: 370 nm and 420 nm. The corresponding emission maxima were 430 and 490 nm, respectively. The A₂-cell fluorophore was very resistant to diffusion caused by the hydration in the histochemical reaction. It was also quite UV-stable, in contrast to the histamine fluorophore which is highly diffusible and UV-labile. The OPT-reactive compounds in rat pineal extracts were analyzed by ion-exchange chromatography. It was possible to establish the presence of OPT-fluorescent compounds not identical with histamine. It is evident that the identity of histamine at histochemically detectable sites has to be confirmed by cytospectrofluorometric and chemical analysis.